cis-Regulatory Sequences and Functions

Multiple regulatory safeguards confine the expression of the GATA factor serpent to the hemocyte primordium within the Drosophila mesoderm

Serpent (srp) encodes a GATA-factor that controls various aspects of embryogenesis in Drosophila, such as fatbody development, gut differentiation and hematopoiesis. During hematopoiesis, srp expression is required in the embryonic head mesoderm and the larval lymph gland, the two known hematopoietic tissues of Drosophila, to obtain mature hemocytes. srp expression in the hemocyte primordium is known to depend on snail and buttonhead, but the regulatory complexity that defines the primordium has not been addressed yet. This study found that srp is sufficient to transform trunk mesoderm into hemocytes. Two disjoint cis-regulatory modules were identified that direct the early expression in the hemocyte primordium and the late expression in mature hemocytes and lymph gland, respectively. During embryonic hematopoiesis, a combination of snail, buttonhead, empty spiracles and even-skipped confines the mesodermal srp expression to the head region. This restriction to the head mesoderm is crucial as ectopic srp in mesodermal precursors interferes with the development of mesodermal derivates and promotes hemocytes and fatbody development. Thus, several genes work in a combined fashion to restrain early srp expression to the head mesoderm in order to prevent expansion of the hemocyte primordium (Spahn, 2013).

Transcriptional Regulation

serpent might act downstream of the anterior and posterior terminal gap gene huckebein (Reuter, 1994).

How does Drosophila mesoderm become subdivided? The process may be illustrated by Bagpipe expression, which is restricted to metameric clusters of cells in the dorsal mesoderm. Under the control of bap, cell clusters develop into midgut visceral mesoderm, whereas cells in segmental portions lacking bap form other mesodermal derivatives. The anterior border of each of the bap patches coincide with the parasegmental borders of the ectoderm. even-skipped transcription marks heart progenitors that will form pericardial cells. Double stainings for BAP and EVE demonstrate that these heart progenitors form between the bap patches at the dorsal crest of the mesoderm. There are also primordia of the fat body in each segment, and these are marked by the expression of serpent. Double staining for SRP and Engrailed show that these cells are at the same anteroposterior positions and lie just ventrolateral to the primordia of the midgut visceral mesoderm (Azpiazu, 1996).

Pair rule genes, active in the ectoderm, seem to regulate segmentation and specification of the visceral mesoderm. Alterations of bap and en are found in mutants of all pair rule genes tested. Because the pair-rule gene products disappear prior to the stage when bap and srp are fully active, their function in regulating these genes must be indirect and involve intermediates. Among the segment polarity genes, both hedgehog and en are required for full activation of bap. These results suggest that hh and en participate in the establishment of the mesodermal posterior (P) domains opposite the posterior domains of the ectoderm. Wingless is synthesized adjacent to the anterior (A) domains. Wingless appears to act negatively on bap and srp, because bap and srp expression is expanded in wg mutant embryos. Thus it appears that ectodermal WG and HH have opposing roles in establishing mesodermal A and P domains (Azpiazu, 1996).

It is clear that pair-rule genes act with the mesoderm to effect its segmentation. This is evident when looking at a residual segmental pattern of bap in wg:hh double or wg:en:hh triple mutant embryos. Thus, wg and en signals provided by ectodermal patches of cells are not sufficient to mediate normal mesoderm segmentation and bap activation. It is likely that the pair-rule genes first establish a prepattern of gene expression within the mesoderm, and later inductive inputs from the ectoderm ensure that the mesodermal and ectodermal parasegments remain in exact register. What genes are regulated by pair-rule genes in the mesoderm? A potential candidate is the paired-domain gene pox meso (see pox neuro), which is expressed in segmental stripes in the early mesoderm. The expression of twist is laid down in a pattern of stripes, with high-protein levels present in the A domains, but this occurs only after the segmental expression of bap has been established (Azpiazu, 1996).

During gastrulation, the Drosophila mesoderm invaginates and forms a single cell layer in close juxtaposition to the overlying ectoderm. Subsequently, particular cell types within the mesoderm are specified along the anteroposterior and dorsoventral axes. The exact developmental pathways that guide the specification of different cell types within the mesoderm are not well understood. The developmental relationship between two mesodermal tissues in the Drosophila embryo, the gonadal mesoderm and the fat body, has been analyzed. Both tissues arise from lateral mesoderm within the even-skipped domain. Whereas in the eve domain of parasegments 10-12 gonadal mesoderm develops from dorsolateral mesoderm and fat body from ventrolateral mesoderm, in parasegments 4-9 only fat body is specified. The cell fate decision between gonadal mesoderm and fat body identity within dorsolateral mesoderm along the anteroposterior axis is determined by the combined actions of genes including abdA, AbdB and srp; while srp promotes fat body development, abdA allows gonadal mesoderm to develop by repressing srp function. Genetic analysis suggests that before stage 10 of embryogenesis, gonadal mesoderm and the fat body have not yet been specified as different cell types, but exist as a common pool of precursor cells requiring the functions of the tin, zfh-1 and cli genes for their development (Moore, 1998).

Targets of Activity

Serpent binds a sequence element found in the larval promoters of all known Alcohol dehydrogenase (Adh) genes. Serpent-binding sites within the D. mulleri and D. melanogaster larval Adh promoters function as positive regulatory elements. In cotransfection experiments, SER functions as a transcriptional activator. SER mRNA is expressed in the embryonic fat body, a tissue that contains high levels of ADH mRNA. The fat body develops from segmentally repeated clusters of mesodermal cells, which later expand and coalesce to form the mature fat body. These observations establish SER as the earliest known fat body precursor marker in the Drosophila embryo (Abel, 1993).

Genes normally expressed in the primordia of the anterior and posterior midgut but not in the hindgut (like caudal and Fasciclin II) are not present in serpent mutants (Reuter, 1994). serpent is responsible for the repression of forkhead in the midgut. The expression of forkhead in the peripheral yolk nuclei is also dependent on serpent (Reuter, 1994).

The fat body precursors are present in serpent mutants. However, the cells do not proliferate and do not rearrange to form the continuous sheet of cells observed in wild-type embryos at late stage 11. Furthermore, the early events of fat body differentiation do not take place in srp mutants. Expression of seven-up, important in fat body development, is not initiated in the mesoderm of srp mutants (Rehorn, 1997).

Hemocytes are cells of mesodermal origin that disperse along migratory pathways in the embryo. The first stage at which they can be identified is late stage 10 in a subpopulation of cells located in the head of the embryo. During the first wave of apoptosis, normally occurring during midembryogenesis, some of them convert to macrophages that engulf and degrade cells undergoing programmed cell death. Glial cell differentiation in Drosophila melanogaster requires the activity of glide (glial cell deficient), also known as glial cells missing (gcm). The role of this gene is to direct the cell fate switch between neurons and glial cells by activating the glial developmental program in multipotent precursor cells of the nervous system. glide/gcm is also expressed and required in the lineage of hemocytes/macrophages, scavenger cells that phagocytose cells undergoing programmed cell death. The earliest glide/gcm expression in the hemocyte lineage can be detected in the head region at the end of the blastoderm stage. By stage 11, glide/gcm expression in the hemocyte lineage decreases, while its expression in glial cells of the peripheral and central nervous systems becomes evident. For glial cells, glide/gcm plays an instructive role in hemocyte differentiation. Interestingly, it has been shown that in the development of the fly adult nervous system the role of scavenger cells is played by glial cells. glide/gcm expression in the hemocyte lineage requires serpent. These data and and evidence regarding on the dual role of glide/gcm indicate that glial cells and hemocytes/macrophages are functionally and molecularly related (Vivancos, 1997).

The yolk protein genes (Yp1 and Yp2) of Drosophila melanogaster are expressed in the fat body tissue and ovarian follicle cells of adult females. A single 12-bp DNA element that activates transcription from the promoters of both Yp genes is tissue specific: it activates transcription of Yp1 and Yp2 reporter genes in follicle cells but has no detectable effect in fat body or other tissues. The sequence of the element consists of two recognition sites for the GATA family of transcription factors. Only dGATAb (serpent) is expressed in ovaries. The single transcript that is detected in ovaries is alternatively spliced or initiated to produce an ovary-specific isoform of the protein. Bacterially expressed serpent binds to the 12-bp element of the Yp promoter (Lossky, 1995).

The GATA motif is a well known positive cis-regulatory element in vertebrates. Experimental evidence is provided for the direct participation of a GATA motif in the expression of the Drosophila antibacterial peptide gene Cecropin A1. A kappaB-like site is necessary for Cecropin A1 gene expression. The Drosophila Rel protein which binds to the kappaB-like site, requires an intact GATA site for maximal Dif-mediated transactivation of the Cecropin A1 gene. A Drosophila blood cell line contains factors binding specifically to the GATA motif of the Cecropin A1 gene. The GATA binding activity is likely to include member(s) of the GATA family of transcriptional regulators. The promoters of several inducible insect immune genes possess GATA sites 0-12 base pairs away from kappaB-like sites in functionally important promoter regions. The serpent gene is expressed both in fat body and hemocytes, and embryos mutatnt for srp lack mature fat body and hemocytes. Like the srp gene, the Cec genes are also expressed in fat body and hemocytes. The overlapping expression pattern of srp and Cec genes makes serpent an interesting candidate for the GATA-binding activity. Clusters of GATA and kappaB sites are also observed in the promoters of two important mammalian immune genes: IL6 and IL3. The consistent proximity of GATA and kappaB sites appears to be a common theme in the immune gene expression of insects and mammals (Kadalayil, 1997).

Two major classes of cells observed within the Drosophila hematopoietic repertoire are plasmatocytes/macrophages and crystal cells. The transcription factor Lz (Lozenge), which resembles human AML1 (acute myeloid leukemia- 1) protein, is necessary for the development of crystal cells during embryonic and larval hematopoiesis. Another transcription factor, Gcm (glial cells missing), is required for plasmatocyte development. Misexpression of Gcm causes crystal cells to be transformed into plasmatocytes. The Drosophila GATA protein Srp (Serpent) is required for both Lz and Gcm expression and is necessary for the development of both classes of hemocytes, whereas Lz and Gcm are required in a lineage-specific manner. Given the similarities of Srp and Lz to mammalian GATA and AML1 proteins, observations in Drosophila are likely to have broad implications for understanding mammalian hematopoiesis and leukemias (Lebestky, 2000).

Hemocytes of the Drosophila embryo are derived from the head mesoderm. The hemocyte precursors express the GATA factor Srp and give rise to two classes of cells: plasmatocytes and crystal cells. Plasmatocytes spread throughout the endolymph and act as macrophages, whereas crystal cells contain crystalline inclusions and are involved in the melanization of pathogenic material in the hemolymph. These cells can be first recognized in the late embryo, where they form a cluster around the proventriculus. Crystal cells are made clearly visible by the Black cell (Bc) mutation, which causes premature melanization of the crystalline inclusions (Lebestky, 2000 and refereces therein).

In larval stages, hemocytes are produced from a separate organ called the lymph gland. Precursors of this gland first appear during embryogenesis in the dorsal mesoderm of the thoracic segments. Later, these precursors migrate dorsally, forming a tight cluster adjacent to the dorsal vessel, the larval circulatory organ. The larval lymph glands form a bilateral chain of cell clusters ('lobes') flanking the dorsal vessel. In the temperature-sensitive allele lzts1, crystal cells develop normally at 25°C. However, crystal cell development is completely blocked at 29°C. Consistent with earlier genetic analysis, crystal cells are missing in lz null mutant alleles. Plasmatocytes develop normally in number and pattern in lz null embryos. Temperature shifts of lzts1;Bc flies show that Lz function during stages 10 to 14 of embryogenesis is essential for crystal cell development. Crystal cells formed in the embryo do not persist into late larvae, and Lz function is continuously required during the late larval stages for further crystal cell development. The time scale for de novo crystal cell development in the larva is about 4.5 hours (Lebestky, 2000).

Lz is first detected in a small cluster of cells within the embryonic head mesoderm in a bilaterally symmetric pattern. Lz expression remains localized in bilateral clusters of 20 to 30 cells within the head mesoderm. At later stages, these crystal cell precursors (CCPs) form a loose cluster around the proventriculus. These cells have smooth, round morphology with large nuclei. The CCPs form a subset of the Srp-expressing hemocyte precursors (Lebestky, 2000).

Colocalization with a mitotic marker suggests that Lz-expressing cells can divide. Interestingly, not all of the daughter cells from these divisions will become crystal cells. This is inferred from the observation that lz-lacZ expression is also seen in a group of plasmatocytes that do not express lz mRNA or Lz protein. The expression of lz-lacZ in these cells is interpreted to be due to the long half-life of beta-galactosidase protein that is left over from the parent cell. This is also observed with additional, independent lz promoter fusions to lacZ. Thus, Lz is expressed in a small subset of hemocyte precursors that may undergo cell division. All crystal cells resulting from these precursors maintain Lz expression. The few daughter cells that will differentiate into plasmatocytes do not express Lz protein (Lebestky, 2000).

In the larval lymph gland, Lz expression is initiated in a small number of cells during the second larval instar. The number of cells expressing Lz steadily increases during the third larval instar, reaching 50 to 100 cells per lobe. Lz-expressing cells are scattered uniformly throughout the large, primary lobe of the lymph gland, whereas the smaller secondary lobes do not express Lz. Similar to the embryonic head mesoderm, all lymph gland cells express Srp, but only a small subset of them express Lz. Interestingly, the Lz-expressing cells appear to down-regulate Srp when compared to the surrounding non-Lz-expressing hemocyte precursors (Lebestky, 2000).

Immunolocalization studies of circulating hemocytes in third-instar larvae suggest that the expression of Lz protein is maintained in circulating crystal cells. Given that crystal cells are missing in lz mutants, this demonstrates an autonomous requirement for Lz in crystal cell development. As observed for embryonic hemocytes, Lz-expressing precursors give rise to all crystal cells and a small subset of plasmatocytes, as evidenced by morphology as well as expression of the plasmatocyte marker Croquemort. However, Lz protein is not observed in any circulating larval plasmatocytes (Lebestky, 2000).

An allele of srp (srpneo45) specifically abolishes Srp expression in embryonic hemocytes. Because this allele also eliminates lz mRNA expression, Srp function is required for the expression of Lz. This finding establishes that srp functions upstream of lz during embryonic hematopoiesis. The lethality of srp precludes the analysis of Lz expression in larval lymph glands of srp mutants. However, as in the embryo, Srp is expressed earlier than Lz in the larval hemocyte precursors, which suggests that srp acts upstream of lz during both developmental stages (Lebestky, 2000).

The transcription factor Gcm promotes glial cell fate, and it also functions downstream of Srp in plasmatocyte differentiation. Lz expression is unaffected in gcm mutants. Gcm expression is initiated in a number of Srp-expressing hemocyte precursors, but Gcm is excluded from the CCPs. Consistent with their cell fate, the small subset of plasmatocytes derived from Lz-expressing progenitors do initiate Gcm expression. Gcm was misexpressed in the CCPs to assess whether exclusion of Gcm from these cells is essential for proper fate determination. This results in the transformation of CCPs into plasmatocytes. The converted cells exhibit morphological characteristics of plasmatocytes and express Croquemort. Moreover, in third-instar larvae, misexpression of Gcm in CCPs prevents the development of all crystal cells. These results suggest that the restricted expression of Gcm is required for the developmental program of embryonic plasmatocytes, and that its misexpression can override Lz-mediated crystal cell differentiation during both embryonic and larval hematopoiesis. The converse experiment of Lz misexpression in the entire hemocyte pool under the control of a heat shock promoter does not convert plasmatocytes into crystal cells. Vertebrate homologs of Gcm have been identified, but any role in hematopoiesis has not been investigated (Lebestky, 2000).

A model of Drosophila hematopoiesis is presented in which a pool of Srp-positive hemocyte precursors gives rise to a large population of Gcm-positive cells and a smaller subpopulation of Lz-positive cells. These results support a genetic hierarchy in which Srp, a Drosophila GATA factor, acts upstream of both Gcm and Lz, two mutually exclusive, lineage-specific transcription factors in hematopoiesis. Although the description of this hierarchy is incomplete in terms of the breadth of molecules involved, it does provide a theoretical framework for understanding how early hematopoietic progenitors in the embryo can differentiate and assume distinct cell fates (Lebestky, 2000).

An ecdysone response unit (EcRU) directs the expression of the Fat body protein 1 (Fbp1) gene in the third instar larval Drosophila fat body. The tissue-specific activity of this regulatory element necessitates the binding of both the ligand-activated EcR/USP ecdysone receptor and GATAb (Serpent). To analyze the role played by GATAb in the regulation of the Fbp1 EcRU activity, the GATA-binding sites GBS1, GBS2 and GBS3 in the Fbp1 EcRU has been replaced with UAS sites for the yeast GAL4 activator and the activity of the mutagenized Fbp1 EcRUs has been tested in transgenic lines, either in the presence or absence of ubiquitously expressed GAL4. GATAb plays two distinguishable roles at the Fbp1 EcRU that contribute to the tissue-specific activity of this regulatory element: (1) GATAb mediates a fat body-specific transcriptional activation and (2) it antagonizes specifically in the fat body a ubiquitous repressor that maintains the Fbp1 EcRU in an inactive state, refractory to activation by GAL4. This repressor has been identified as AEF-1, a factor shown to be involved in the regulation of the Drosophila Adh and yp1-yp2 genes. These results show that, for a functional dissection of complex promoter-dependent regulatory pathways, the replacement of specific regulatory target sites by UAS GAL4 binding sites is a powerful alternative to the widely used disruption approach (Brodu, 2001).

Of the three GATAb binding sites present in the Fbp1 EcRU, only GBS1 is crucially required for the activity of the Fbp1 EcRU in the third larval instar fat body in response to ecdysone, whereas GBS2 and GBS3 seemed to be dispensable. Although this result indicates that binding of GATAb to the EcRU is essential, it gives no clue to the function of this factor other than that of transactivation. Because the replacement of GBS sites by a UAS site allows a strong transcriptional activator to be targeted to the EcRU, it has become feasible to examine whether GATAb is still required for the activity of the EcRU when GAL4 is present. The clear-cut patterns of expression of four constructs provide an unambiguous answer to this question. The AE[GBS1m-UAS2-3] and AE[UAS1-GBS3m] constructs, where the GBS1 and GBS3 sites, respectively, are inactivated, show a total lack of expression in the GAL4daG32 context, while the AE[UAS2-3] and AE[UAS1-GBS2m] constructs, where one of these GBS sites remains intact, exhibit full expression in the same context. This provides a strong argument in support of the hypothesis that activation of the Fbp1 EcRU by GAL4 crucially requires the binding of GATAb to at least one GBS site and indicates that this factor is specifically involved in a competence step that makes the EcRU responsive to transcriptional activators in the third larval instar fat body. Remarkably, the substitution approach reveals a functional redundancy of GBS1 and GBS3 in mediating this competence. In contrast, GBS2 does not appear to be able to support the same functional role. The 5'GATT3' core sequence of this site differs from the canonical 5'GATA3' core target sequence found in both GBS1 and GBS3 and the GATAb binding affinity for GBS2 is lower than that for GBS1 and GBS3. Together, these data suggest that the apparent absence of any functional role for GBS2 in the Fbp1 EcRU activity is related to its lower in vivo affinity for GATAb (Brodu, 2001).

What are the mechanisms involved in the competence function of GATAb, as revealed by the UAS substitution approach? Numerous studies have shown that the yeast transcription factor GAL4 is able to activate reporter transgenic constructs under the control of UAS sites in all tissues, including those in which GATAb is not expressed. These data make it very unlikely that one of GATAb functions is to specifically potentiate the transactivating activity of GAL4 in the fat body tissue. The restriction of the expression of the AE[UAS1], AE[UAS2-3] and AE[UAS1-GBS2m] constructs to the fat body in a GAL4daG32 genetic context provides a strong argument in favor of the idea that specific sequences in these constructs target a potent ubiquitous repressor of GAL4 activity, which is antagonized solely in this tissue by means of a GATAb-dependent mechanism. The observation that the 5UAS-Fbp1-lacZ control construct is expressed throughout development in most tissues when crossed in the GAL4daG32 animals indicates that the Fbp1 minimal promoter is fully responsive to GAL4 and does not contain any such repressor binding sequences. Similarly, the strong and fat-body specific expression of the AE[UAS1-UASEBS] construct excludes the notion that the binding site for the EcR-USP receptor plays this role. In contrast the results clearly demonstrate that element A mediates the binding of the strong repressor AEF-1 (Brodu, 2001).

Two mechanisms of gene repression by transcriptional interference have been characterized so far for AEF-1. It has been shown that the binding of AEF-1 to the Adult Adh enhancer negatively regulates the Adh gene by interfering with the binding of an activator of the C/EBP family to an overlapping site. A similar binding interference between AEF-1, C/EBP and the female-specific Doublesex protein was proposed for the downregulation of the yolk protein genes Yp1 and Yp2. AEF-1 also binds to the initiator region (Inr) of the Adh proximal promoter and represses transcription by a distinct mechanism thought to involve steric interference with the binding of general transcription factors. In contrast, the finding that AEF-1 is able to block the activation of the Fbp1 EcRU by GAL4 targeted to a site more than 50 bp downstream from the AEF-1 site, provides evidence that AEF-1 has yet another function, which is to repress enhancers at a distance (Brodu, 2001).

Transcriptional repressors have been characterized by their range of action on promoters and enhancers. Short-range repressors, including Snail, Knirps and Krüppel, interact over distances of 50-150 bp to inhibit, or quench, either transcriptional activators or the basal transcription complex. These repressors share a conserved PXDLSXK sequence motif, responsible for interaction with the corepressor dCtBP. In contrast, long-range repressors, including Dorsal and Hairy, act over distances of several kilobases to silence basal promoters and interact with the corepressor Groucho through a conserved WRPW motif. The data presented here suggest that AEF-1 belongs to the short-range repressor family. Whether AEF-1 is also able to act as a long-range repressor requires additional experiments (Brodu, 2001).

The lack of PXDLSXK or WRPW motives in AEF-1 suggests that it mediates repression through an interaction with corepressors other than dCtBP and Groucho. Evidence that histone deacetylation plays a role in gene silencing has accumulated in recent years. It has been shown in particular that the histone deacetylase Rpd3, and the Sin3A (see Drosophila Sin3A) and SMRT/NcoR proteins are part of a corepressor complex of mammalian transcriptional repressors. Similarly, Rpd3 and the Drosophila SMRT homolog SMRTER have been to interact with Groucho and the unliganded EcR/USP ecdysone receptor. In this context, a possible link between AEF-1 and complexes displaying a histone-deacetylase activity deserves investigation (Brodu, 2001).

Innate immunity in Drosophila is characterized by the inducible expression of antimicrobial peptides. An investigation was carried out of the development and regulation of immune responsiveness in Drosophila embryos after infection. Immune competence, as monitored by the induction of Cecropin A1-lacZ constructs, is observed first in the embryonic yolk. This observation suggests that the yolk plays an important role in the humoral immune response of the developing embryo by synthesizing antimicrobial peptides. Around midembryogenesis, the response in the yolk is diminished. Simultaneously, Cecropin expression becomes inducible in a large number of cells in the epidermis, demonstrating that late-stage embryos can synthesize their own antibiotics in the epidermis. This production likely serves to provide the hatching larva with an active antimicrobial barrier and protection against systemic infections. Cecropin expression in the yolk requires the presence of a GATA site in the promoter as well as the involvement of the GATA-binding transcription factor Serpent. In contrast, neither the GATA site nor Serpent are necessary for Cecropin expression in the epidermis. Thus, the inducible immune responses in the yolk and in the epidermis can be uncoupled and call for distinct sets of transcription factors. The data suggest that Serpent is involved in the distinction between a systemic response in the yolk/fat body and a local immune response in epithelial cells. In addition, the present study shows that signal transduction pathways controlling innate and epithelial defense reactions can be dissected genetically in Drosophila embryos (Tingvall, 2001).

A complete understanding of the function of the embryonic yolk nuclei (vitellophages) in Drosophila has remained elusive. Studies of the ultrastructure of the vitellophages' cytoplasm reveal large amounts of endoplasmic reticulum and free ribosomes, among other organelles, indicating a high level of differentiation and intense protein synthesis. Another protein linked to immune function in insects, the hemolin protein of Hyalophora cecropia, is expressed constitutively in the yolk during oogenesis and embryogenesis, suggesting that a number of immune-related proteins are expressed in the embryonic yolk. The data suggest that the yolk cell serves a very important function in defending the embryo against microbial infections by the rapid production of antimicrobial peptides (Tingvall, 2001).

The synthesized antimicrobial peptides, which contain a signal peptide, probably can be exported from the yolk cell to the embryo hemocoel and possibly through the amnioserosa into the perivitelline fluid also, in which they can attack invading organisms. The data also indicate that the activation of an immune response requires contact between microbial substances and components present in the perivitelline fluid or in the embryo, because the presence of bacteria on the surface of embryos with an intact vitelline membrane does not mount a response. Therefore, the chorion and the vitelline membrane may provide a physical barrier to infection (Tingvall, 2001).

What then may be the normal route of infection in nature? During fertilization, the seminal fluid may be contaminated with bacteria that can enter the egg together with the sperm. The seminal receptacle and spermathecae of the female as well as the reproductive organs of the male are sites of constitutive synthesis of antimicrobial factors, indicating that it is crucial for high reproductive efficiency to minimize bacterial contamination of the seminal fluid. Another threat for the embryo is infection with maternally transmitted endocellular bacteria such as Wolbachia of the Rickettsial family. This microorganism is transmitted vertically from the reproductive organs of the insect female to the egg, from which it migrates to the germ cells laid down in the embryo. Eggs laid by infected Drosophila females contain bacteria scattered in the yolk region and in later stages of development, bacteria are distributed throughout the somatic and germ-line tissues. The immune response is active in the yolk, in which Wolbachia first appear in the developing embryo (Tingvall, 2001).

Expression of CecA1 in the embryonic yolk is inducible only during a relatively narrow time window. The onset of expression probably relies on the de novo synthesis of crucial factors in the zygotic embryo. A probable explanation for the sharp decline in expression about 12 h AEL is that microbial substances or transmitted signals cannot reach the yolk and its nuclei after the time point of dorsal and midgut closure. The data suggest that signals could be transmitted over the amnioserosa, which is a thin extraembryonic membrane that covers the dorsal side of the embryo during gastrulation. This route of signal transmission most likely is blocked after dorsal closure when the amnioserosa is no longer in direct contact with the perivitelline fluid (Tingvall, 2001).

Surprisingly, CecA1 expression is not evident in the embryonic fat body or hemocytes, although these tissues are sites of high-level expression in postembryonic stages, suggesting that important factors are limiting. Instead, the CecA1 gene was inducible in the epidermis after 12 AEL. The results indicate that microbial substances present in the perivitelline fluid can activate the epidermal cells directly during embryogenesis and that in the absence of a hard cuticle, the signal reaches numerous epidermal cells. This result suggests that the epidermal cells express transmembrane receptors that respond to the presence of microbial products in the perivitelline fluid and transduce the signal to the nucleus. Interestingly, the Drosophila transmembrane receptor Toll is expressed in all cells throughout the embryonic epidermis. Therefore, Toll together with other Toll-like receptors are possible candidates for being mediators of the immune response in embryonic epidermis. The development of an epidermal defense probably serves to protect the embryo against infection during the late stages of embryogenesis and to provide the hatching larva with an inducible immune system in the epidermis underlying the larval cuticle (Tingvall, 2001).

GATA factors play an essential role in endodermal specification in both protostomes and deuterostomes. In Drosophila, the GATA factor gene serpent (srp) is critical for differentiation of the endoderm. However, the expression of srp disappears around stage 11, which is much earlier than overt differentiation occurs in the midgut, an entirely endodermal organ. Another endoderm-specific Drosophila GATA factor gene, GATAe, has been identified. Expression of GATAe is first detected at stage 8 in the endoderm , and its expression continues in the endodermal midgut throughout the life cycle. srp is required for expression of GATAe, and misexpression of srp resulted in ectopic GATAe expression. Embryos that either lack GATAe or have been injected with double-stranded RNA (dsRNA) corresponding to GATAe fail to express marker genes that are characteristic of differentiated midgut. Conversely, overexpression of GATAe induces ectopic expression of endodermal markers even in the absence of srp activity. Transfection of the GATAe cDNA also induces endodermal markers in Drosophila S2 cells. These studies provide an outline of the genetic pathway that establishes the endoderm in Drosophila. This pathway is triggered by sequential signaling through the maternal torso gene, a terminal gap gene, huckebein (hkb), and finally, two GATA factor genes, srp and GATAe (Okumura, 2005).

serpent is required for endodermal development in Drosophila. srp is first expressed at the cellular blastoderm stage in yolk cells, and in prospective regions of the endoderm, amnioserosa, and hemocyte primordium. Endodermal expression of srp disappears by stages 10-11. Embryos lacking srp activity fail to develop endoderm; instead, the prospective endodermal region develops into the ectodermal hindgut. Since srp is expressed in the endoderm earlier than GATAe is expressed, activation of GATAe expression by srp was examined. Expression of GATAe in the prospective endoderm region is abolished in the srp mutant (srp2/srp2), whereas GATAe expression in the Malpighian tubules is not affected. Conversely, ubiquitously misexpressed srp causes strong ectopic expression of GATAe in the foregut, and in the hindgut. Note that the foregut and hindgut arise immediately anterior and posterior to the endoderm, respectively. This ectopic expression pattern is transient. During embryogenesis, GATAe is also induced ectopically in the salivary gland and segmentally in the ventral nerve cord. These results strongly suggest that srp activates GATAe in the endoderm. fork head (fkh) is expressed throughout the prospective gut, and the gut primordia degenerate during germband retraction in fkh mutants. However, GATAe expression is not affected in the fkh mutant (fkhXT6/fkhXT6) (Okumura, 2005).

Hand is a direct target of Tinman and GATA factors during Drosophila cardiogenesis and hematopoiesis

The Hand gene family encodes highly conserved basic helix-loop-helix (bHLH) transcription factors that play crucial roles in cardiac and vascular development in vertebrates. In Drosophila, a single Hand gene is expressed in the three major cell types that comprise the circulatory system: cardioblasts, pericardial nephrocytes and lymph gland hematopoietic progenitors. Drosophila Hand functions as a potent transcriptional activator, and converting it into a repressor blocks heart and lymph gland formation. Disruption of Hand function by homologous recombination also results in profound cardiac defects that include hypoplastic myocardium and a deficiency of pericardial and lymph gland hematopoietic cells, accompanied by cardiac apoptosis. Targeted expression of Hand in the heart completely rescues the lethality of Hand mutants, and cardiac expression of a human HAND gene, or the caspase inhibitor P35, partially rescues the cardiac and lymph gland phenotypes. These findings demonstrate evolutionarily conserved functions of HAND transcription factors in Drosophila and mammalian cardiogenesis, and reveal a previously unrecognized requirement of Hand genes in hematopoiesis (Han, 2006).

The existence of hemangioblasts, which serve as common progenitors for hematopoietic cells and cardioblasts, has suggested a molecular link between cardiogenesis and hematopoiesis in Drosophila. However, the molecular mediators that might link hematopoiesis and cardiogenesis remain unknown. This study shows that the highly conserved bHLH transcription factor Hand is expressed in cardioblasts, pericardial nephrocytes and hematopoietic progenitors. The homeodomain protein Tinman and the GATA factors Pannier and Serpent directly activate Hand in these cell types through a minimal enhancer, which is necessary and sufficient to drive Hand expression in these different cell types. Hand is activated by Tinman and Pannier in cardioblasts and pericardial nephrocytes, and by Serpent in hematopoietic progenitors in the lymph gland. These findings place Hand at a nexus of the transcriptional networks that govern cardiogenesis and hematopoiesis, and indicate that the transcriptional pathways involved in development of the cardiovascular, excretory and hematopoietic systems may be more closely related than previously appreciated (Han, 2005).

To search for cis-regulatory elements capable of conferring the specific expression pattern of Hand in cardioblasts, pericardial nephrocytes and lymph gland hematopoietic progenitors, a series of reporter genes were generated containing lacZ and the hsp70 basal promoter linked to genomic fragments within a 13 kb genomic region encompassing the gene, and reporter gene expression was examined in transgenic embryos. A 513 bp minimal enhancer was identified referred to as Hand cardiac and hematopoietic (HCH) enhancer, between exons 3 and 4 of the Hand gene. HCH is both necessary and sufficient to direct lacZ expression in the entire embryonic heart and lymph gland in a pattern identical to that of the endogenous Hand gene. Further deletions of this enhancer caused either a partial or complete loss of activity. The 513 bp HCH enhancer showed the same expression pattern in the heart and lymph gland as larger genomic fragments that were positive for enhancer activity. It is concluded that this enhancer fully recapitulates the temporal and spatial expression pattern of Hand transcription in the distinct cell types derived from the cardiogenic region (Han, 2005).

The homeobox protein Tinman is essential for the formation of the cardiac mesoderm, from which the heart and blood progenitors arise. However, its potential late functions remain unknown. It is believed that Tinman is not required for the entirety of heart development in flies, because it is not maintained in all the cardiac cells at late stages. The data reveal at least one function for the late-embryonic Tinman expression, which is to maintain Hand expression. The fact that ectopic Tinman can turn on Hand expression dramatically in the somatic muscles is striking and suggests the existence of a Tinman-co-factor in muscle cells that can cooperate with Tinman to activate Hand expression; this co-factor would not be expected to be expressed in pericardial cells or the lymph gland. This co-factor should also be expressed in Drosophila S2 cells, since transfected Tinman can increase activity of the HCH enhancer in S2 cells by more than 100-fold. The generally reduced activity of the HCH enhancer that results from mutation of the Tinman-binding sites also suggests that Tinman activity is required to fully activate the Hand enhancer (Han, 2005).

Although Pannier and Serpent bind to the same consensus sites, these GATA factors produce distinct phenotypes when overexpressed in the mesoderm. Ectopic Pannier induces cardiogenesis, shown by the extra number of cardioblasts and pericardial nephrocytes, but does not affect the lymph gland hematopoietic progenitors. Ectopic Serpent, however, induces ectopic lymph gland hematopoietic progenitors, but reduces the number of cardioblasts and pericardial cells. Interestingly, pericardial cells with ectopic Serpent expression have a tendency to form cell clusters such as the lymph gland progenitors, suggesting a partial cell fate transformation. These results suggest that Pannier functions as a cardiogenic factor, whereas Serpent functions as a hematopoietic factor. Although both can activate Hand expression, Pannier and Serpent activate the HCH enhancer in different cell types. This assumption is also supported by the specific expression pattern of Serpent and Pannier in late embryos. Serpent is detected specifically in the lymph gland hematopoietic progenitors but not in any cardiac cells. Pannier expression in the cardiogenic region of late embryos is not clear because of the interference by the high level Pannier expression from the overlaying ectoderm. However, the lymph gland was examined in late stage embryos and no Pannier expression was detected in these cells. Together with the evidence from loss-of-function and gain-of-function experiments with Serpent, it is concluded that the HCH-5G-GFP transgene is not expressed in the lymph gland because Serpent could not bind to the mutant enhancer in the lymph gland cells; whereas the lack of HCH-5G-GFP expression in cardiac cells is due to the inability of Pannier to bind the mutant enhancer in these cardiac cells (Han, 2005).

Since tin and pnr are not expressed in all the cardiac cells of late stage embryos but the Hand-GFP transgene is expressed in these cells, it is likely that additional factors control Hand expression in the heart. One group of candidates is the T-box family. Since Doc1, Doc2 and Doc3 genes (Drosophila orthologs to vertebrate Tbx5) are expressed in the Svp-positive cardioblasts where tin is not expressed, but H15 and midline (Drosophila orthologs to vertebrate Tbx-11) are expressed in most of the cardiac cells in late embryos, it is likely that the T-box genes activate Hand expression in cells that do not express tin and pannier. However, the enhancer lacking GATA and Tinman sites has no activity, indicating that the additional factors that may activate Hand expression in the heart and lymph gland also requires these crucial Tinman and GATA sites, probably through protein interaction between Tinman and the GATA factors (Han, 2005).

In mammals, the adult hematopoietic system originates from the yolk sac and the intra-embryonic aorta-gonad-mesonephros (AGM) region. The AGM region is derived from the mesodermal germ layer of the embryo in close association with the vasculature. Indeed, the idea of the hemangioblast, a common mesodermal precursor cell for the hematopoietic and endothelial lineages, was proposed nearly 100 years ago without clear in vivo evidence. Recently, this idea was substantiated by the identification of a single progenitor cell that can divide into a hematopoietic progenitor cell in the lymph gland and a cardioblast cell in the dorsal vessel in Drosophila (Mandal, 2004). In addition to providing the first evidence for the existence of the hemangioblast, this finding also suggested a close relationship between the Drosophila cardiac mesoderm, which gives rise to cardioblasts, pericardial nephrocytes and pre-hemocytes, and the mammalian cardiogenic and AGM region, which gives rise to the vasculature (including cardiomyocytes), the excretory systems (including nephrocytes) as well as adult hematopoietic stem cells. In fact, in both Drosophila and mammals, the specification of the cardiogenic and AGM region requires the input of Bmp, Wnt and Fgf signaling. In addition to the conserved role of the NK and GATA factors, GATA co-factors (U-shaped in Drosophila and Fog in mice) also play important roles in cardiogenesis and hematopoiesis in both Drosophila and mammals. Recent studies have shown that the Notch pathway is required for both cardiogenic and hematopoietic progenitor specification in Drosophila. It is likely that Notch also plays an important role in mammalian hematopoiesis (Han, 2005).

This study found that Drosophila Hand is expressed in cardioblasts, pericardial nephrocytes and pre-hemocytes, and is directly regulated by conserved transcription factors (NK and GATA factors) that control both cardiogenesis and hematopoiesis. The bHLH transcription factor Hand is highly conserved in both protein sequence and expression pattern in almost all organisms that have a cardiovascular system. In mammals, Hand1 is expressed at high levels in the lateral plate mesoderm, from which the cardiogenic region and the AGM region arise, in E9.5 mouse embryos. Functional studies of Hand1 and Hand2 using knockout mice have demonstrated the essential role of Hand genes during cardiogenesis, whereas the functional analysis of Hand genes during vertebrate hematopoiesis has not yet been explored. It will be interesting to determine whether mammalian Hand genes are also regulated in the AGM region by GATA1, GATA2 and GAT3 (vertebrate orthologs to Drosophila Serpent), and whether they play a role in mammalian hematopoiesis (Han, 2005).

In summary, this study places Hand at a pivotal point to link the transcriptional networks that govern cardiogenesis and hematopoiesis. Since the Hand gene family encodes highly conserved bHLH transcription factors expressed in the cardiogenic region of widely divergent vertebrates and probably in the AGM region in mouse, these findings open an avenue for further exploration of the conserved transcriptional networks that govern both cardiogenesis and hematopoiesis, by studying the regulation and functions of Hand genes in vertebrate model systems (Han, 2005).

Serpent, suppressor of hairless and U-shaped are crucial regulators of hedgehog niche expression and prohemocyte maintenance during Drosophila larval hematopoiesis

The lymph gland is a specialized organ for hematopoiesis, utilized during larval development in Drosophila. This tissue is composed of distinct cellular domains populated by blood cell progenitors (the medullary zone), niche cells that regulate the choice between progenitor quiescence and hemocyte differentiation [the posterior signaling center (PSC)], and mature blood cells of distinct lineages (the cortical zone). Cells of the PSC express the Hedgehog (Hh) signaling molecule, which instructs cells within the neighboring medullary zone to maintain a hematopoietic precursor state while preventing hemocyte differentiation. As a means to understand the regulatory mechanisms controlling Hh production, a PSC-active transcriptional enhancer was characterized that drives hh expression in supportive niche cells. The findings indicate that a combination of positive and negative transcriptional inputs program the precise PSC expression of the instructive Hh signal. The GATA factor Serpent (Srp) is essential for hh activation in niche cells, whereas the Suppressor of Hairless [Su(H)] and U-shaped (Ush) transcriptional regulators prevent hh expression in blood cell progenitors and differentiated hemocytes. Furthermore, Srp function is required for the proper differentiation of niche cells. Phenotypic analyses also indicated that the normal activity of all three transcriptional regulators is essential for maintaining the progenitor population and preventing premature hemocyte differentiation. Together, these studies provide mechanistic insights into hh transcriptional regulation in hematopoietic progenitor niche cells, and demonstrate the requirement of the Srp, Su(H) and Ush proteins in the control of niche cell differentiation and blood cell precursor maintenance (Tokusumi, 2010).

The lymph gland hematopoietic organ is formed near the end of embryogenesis from two clusters of cells derived from anterior cardiogenic mesoderm (Crozatier, 2004; Mandal, 2004). About 20 pairs of hemangioblast-like cells give rise to three distinct lineages that will form the lymph glands and anterior part of the dorsal vessel. Notch (N) pathway signaling serves as the genetic switch that differentially programs these progenitors towards cell fates that generate the lymph glands (blood lineage), heart tube (vascular lineage), or heart tube-associated pericardial cells (nephrocytic lineage). An essential requirement has also been proven for Tailup (Islet1) in lymph gland formation, in which it functions as an early-acting regulator of serpent, odd-skipped and Hand hematopoietic transcription factor gene expression (Tokusumi, 2010).

By the end of the third larval instar, each anterior lymph gland is composed of three morphologically and molecularly distinct regions (Jung, 2005). The posterior signaling center (PSC) is a cellular domain formed during late embryogenesis due to the specification function of the homeotic gene Antennapedia (Antp) (Mandal, 2007) and the maintenance function of Collier, the Drosophila ortholog of the vertebrate transcription factor early B-cell factor. PSC cells selectively express the Hedgehog (Hh) and Serrate (Ser) signaling molecules and extend numerous thin filopodia into the neighboring medullary zone. This latter lymph gland domain is populated by undifferentiated and slowly proliferating blood cell progenitors (Mandal, 2007). Prohemocytes within the medullary zone express the Hh receptor Patched (Ptc) and the Hh pathway transcriptional effector Cubitus interruptus (Ci). Medullary zone cells also express components of the Jak/Stat signaling pathway. By contrast, the third lymph gland domain -- the cortical zone -- solely contains differentiating and mature hemocytes, such as plasmatocytes and crystal cells. Upon wasp parasitization, or in certain altered genetic backgrounds, lamellocytes will also appear in the cortical zone as a third type of differentiated hemocyte (Tokusumi, 2010).

Two independent studies have provided compelling data to support the contention that the PSC functions as a hematopoietic progenitor niche within the lymph gland, with this cellular domain being essential for maintaining normal hemocyte homeostasis (Krzemien, 2007; Mandal, 2007). These investigations showed that communication between the PSC and prohemocytes present in the medullary zone is crucial for the preservation of the progenitor population and to prevent these cells from becoming abnormally programmed to differentiate into mature hemocytes. Seminal findings from these studies can be summarized as follows: Col expression must be restricted to the PSC by the localized expression of Ser; Hh must be expressed selectively in the PSC, coupled with the non-autonomous activation of the Hh signaling pathway in prohemocytes of the medullary zone; and the PSC triggers activation of the Jak/Stat pathway within cells of the medullary zone. With the perturbation of any of these molecular events, the precursor population of the medullary zone is lost owing to the premature differentiation of hemocytes, which swell the cortical zone. Although the exact interrelationship of Ser, Hh and Jak/Stat signaling within the lymph gland is currently unknown, the cytoplasmic extensions emanating from PSC cells might facilitate instructive signaling between these niche cells and hematopoietic progenitors present in the medullary zone (Krzemien, 2007; Mandal, 2007). A more recent study showed that components of the Wingless (Wg) signaling pathway are expressed in the stem-like prohemocytes to reciprocally regulate the proliferation and maintenance of cells within the supportive PSC niche (Sinenko, 2009). The cellular organization and molecular signaling of the Drosophila lymph gland are remarkably similar to those of the hematopoietic stem cell niches of vertebrate animals, including several mammals (Tokusumi, 2010 and references therein).

Through detailed molecular and gene expression analyses this study has identified the PSC-active transcriptional enhancer within hh intron 1 and delimited its location to a minimal 190 bp region. The hh enhancer-GFP transgene faithfully recapitulates the niche cell expression of Hh derived from the endogenous gene, as double-labeling experiments with the GFP marker and Antp or Hh show a clear co-expression in PSC domain cells. Appropriately, GFP expression is not detected in Antp loss-of-function or TCFDN genetic backgrounds, which culminate in an absence of niche cells from the lymph gland. The hematopoietic GATA factor Srp serves as a positive activator of hh PSC expression, as mutation of two evolutionarily conserved GATA elements in the enhancer abrogates its function and Srp functional knockdown via srp RNAi results in hh enhancer-GFP transgene inactivity and the absence of Hh protein expression. An additional intriguing phenotype was observed in lymph glands expressing the srp RNAi transgene, that being a strong reduction in the number of filopodial extensions emerging from cells of the PSC. This phenotype suggests a functional role for Srp in the correct differentiation of niche cells, via a requirement for normal Hh presentation from these cells and/or the transcriptional regulation by Srp of additional genes needed for the formation of filopodia (Tokusumi, 2010).

As Srp accumulates in all cells of the lymph gland, a question arose as to how hh expression is restricted to cells of the PSC. This paradox could be explained by a mechanism in which hh expression is also under some means of negative transcriptional control in non-PSC cells of the lymph gland. This possibility proved to be correct, with the analyses identifying two negative regulators of hh lymph gland expression. The first is Su(H). Mutation of the evolutionarily conserved GTGGGAA element, a predicted recognition sequence for this transcriptional repressor, resulted in an expanded activity of the hh PSC enhancer-GFP transgene; that is, the de novo appearance of GFP was observed in prohemocytes of the medullary zone. Likewise, ectopic medullary zone expression of the wild-type PSC enhancer-GFP transgene and of Hh protein was seen in lymph glands mutant for Su(H). These findings, coupled with the detection of Su(H) in blood cell progenitors, strongly implicate this factor as a transcriptional repressor of the hh PSC enhancer, restricting its expression to niche cells (Tokusumi, 2010).

Additional studies identified Ush as a second negative regulator of hh expression. Ush is expressed in most cells of the lymph gland, with the exception of those cells resident within the PSC domain. Previous research demonstrated that ush expression in the lymph gland is under the positive control of both Srp. Why Ush protein fails to be expressed in the PSC remains to be determined. Forced expression of ush in niche cells resulted in inactivation of the hh PSC enhancer and reduced the formation of filopodia. It was hypothesized that Ush might be forming an inhibitory complex with the SrpNC protein, changing Srp from a positive transcriptional activator to a negative regulator of hh lymph gland expression. Such a mechanism has been demonstrated previously in the negative regulation by Ush of crystal cell lineage commitment. The expansion of wild-type hh enhancer-GFP transgene and Hh protein expression to prohemocytes within the medullary zone and to differentiated hemocytes within the cortical zone in lymph glands mutant for ush is also supportive of Ush functioning as a negative regulator of hh expression (Tokusumi, 2010).

Bringing these results together, a model can be proposed for the regulatory events that culminate in the precise expression of the vital Hh signaling molecule in niche cells. Srp is a direct transcriptional activator of hh in the lymph gland and Hh protein is detected in niche cells due to this activity. hh expression is inhibited in prohemocytes of the medullary zone by Su(H) action, while a repressive SrpNC-Ush transcriptional complex prevents Srp from activating hh expression in prohemocytes and in differentiated hemocytes of the medullary zone and cortical zone. Together, these positive and negative modes of regulation would allow for the niche cell-specific expression of Hh and facilitate the localized presentation of this crucial signaling molecule to neighboring hematopoietic progenitors (Tokusumi, 2010).

The identification of Srp and Su(H) as key regulators of Hh expression in the larval hematopoietic organ prompted an investigation into the functional requirement of these proteins in the control of blood cell homeostasis. Since Srp knockdown by RNAi leads to an absence of the crucial Hh signal, it was not surprising to find that normal Srp function is required for prohemocyte maintenance and the control of hemocyte differentiation within the lymph gland; that is, a severe reduction of Ptc-positive hematopoietic progenitors and a strong increase in differentiated plasmatocytes and crystal cells was observed in srp mutant tissue (Tokusumi, 2010).

Likewise, Ptc-positive prohemocytes were lost and large numbers of plasmatocytes were prematurely formed in Su(H) mutant lymph glands. This disruption of prohemocyte maintenance occurred even though Hh protein expression was expanded throughout the medullary zone. This raised the question as to why expanded Hh protein and possible Hh pathway activation did not increase the progenitor population in Su(H) mutant lymph glands, instead of the observed loss of prohemocytes and appearance of differentiated plasmatocytes. One explanation might be that the PSC niche is not expanded in Su(H) mutant lymph glands and Hh might only function in promoting blood cell precursor maintenance within the context of the highly ordered progenitor-niche microenvironment. It has been hypothesized that the filopodial extensions that emanate from differentiated niche cells are crucial for Hh signal transduction from the PSC to progenitor cells of the medullary zone. The possibility exists that ectopic Hh protein, which is not produced or presented by niche cells, is unable to positively regulate prohemocyte homeostasis. An experimental result consistent with this hypothesis is that expression of UAS-hh under the control of the medullary zone-specific tepIV-Gal4 driver failed to expand the blood cell progenitor population. A second possibility is that the Hh pathway transcriptional effector Ci might require the co-function of Su(H) in its control of prohemocyte maintenance. This model would predict that, in the absence of Su(H) function, Hh signaling would be less (or non) effective in controlling the genetic and cellular events needed for the maintenance of the prohemocyte state. Third, Su(H) might regulate additional target genes, the expression (or repression) of which is crucial for normal blood cell precursor maintenance and the prevention of premature hemocyte differentiation. Finally, it cannot be ruled out that the expression of ectopic Hh in medullary zone cells, in the context of the adverse effects of Su(H) loss of function in these cells, culminates in the disruption of normal Hh pathway signaling due to an unforeseen dominant-negative effect (Tokusumi, 2010).

In summary, these findings add significantly to knowledge of hematopoietic transcription factors that function to control stem-like progenitor maintenance and blood cell differentiation in the lymph gland. An additional conclusion from these studies is that the hh enhancer-GFP transgene can serve as a beneficial reagent to identify and characterize genes and physiological conditions that control the cellular organization of the hematopoietic progenitor-niche cell microenvironment. RNAi-based genetic screens could be undertaken using this high-precision marker to determine signaling pathways and/or environmental stress conditions that might alter niche cell number and function, leading to an alteration in hematopoietic progenitor maintenance coupled with the robust production of differentiated blood cells. Much remains to be determined about the regulated control of these critical hematopoietic changes and their likely relevance to hematopoietic stem cell-niche interactions in mammals (Tokusumi, 2010).

A genome-wide RNA interference screen identifies a differential role of the mediator CDK8 module subunits for GATA/ RUNX-activated transcription in Drosophila

Transcription factors of the RUNX and GATA families play key roles in the control of cell fate choice and differentiation, notably in the hematopoietic system. During Drosophila hematopoiesis, the RUNX factor Lozenge and the GATA factor Serpent cooperate to induce crystal cell differentiation. This study used Serpent/Lozenge-activated transcription as a paradigm to identify modulators of GATA/RUNX activity by a genome-wide RNA interference screen in cultured Drosophila blood cells. Among the 129 factors identified, several belong to the Mediator complex. Mediator is organized in three modules plus a regulatory "CDK8 module," composed of Med12, Med13, CycC, and Cdk8, which has long been thought to behave as a single functional entity. Interestingly, the data demonstrate that Med12 and Med13 but not CycC or Cdk8 are essential for Serpent/Lozenge-induced transactivation in cell culture. Furthermore, in vivo analysis of crystal cell development show that, while the four CDK8 module subunits control the emergence and the proliferation of this lineage, only Med12 and Med13 regulate its differentiation. It is thus proposed that Med12/Med13 acts as a coactivator for Serpent/Lozenge during crystal cell differentiation independently of CycC/Cdk8. More generally, it is suggested that the set of conserved factors identified in this study may regulate GATA/RUNX activity in mammals (Gobert, 2010).

During development, a combination of general and lineage-specific transcription factors integrate different regulatory inputs at the transcriptional levels to unfold the proper gene expression program. The identification of the complete panel of genes that participate in the regulation of the activity of these transcription factors is critical to understand the fine-tuning of transcription that underlies cellular differentiation. In this study, a genome-wide RNAi screen was conducted to uncover regulators of the activity of the GATA/RUNX complex Srp/Lz. This approach highlighted the function of the Mediator complex in Srp/Lz-induced transcriptional activation. Moreover, it was found that, within the Mediator CDK8 module, Med12 and Med13 act independently of CycC and Cdk8 to promote Srp/Lz-dependent transactivation and blood cell differentiation (Gobert, 2010).

The activity of GATA and RUNX transcription factors has been shown to be regulated by interaction with several factors, such as the coactivator CBP/p300 or the corepressors HDAC and Sin3A. However, proper transcriptional regulation relies on the coordinated action of several transcription factors binding a particular cis-regulatory element. Notably, GATA and RUNX factor have been shown to cooperate in both mammals and Drosophila to regulate the expression of specific target genes. Hence, this study used Srp/Lz cooperation as a paradigm to identify putative coregulators of GATA/RUNX activity. Among the genes that were identified, five (CKD9, SIN3A, MED1, enok homolog MYST3/MOZ, and pnt homolog ETS1) have been linked previously to GATA and/or RUNX activity in mammals, and four (Pcf11, CtBP, med13, and Sin3A) have been linked to crystal cell development in flies. This brings strong support to the conclusion that the cell-based assay is suitable to identify genuine modulators of Srp/Lz activity and, more generally, of GATA/RUNX factors. However, further work will be required to discriminate between factors affecting GATA/RUNX interplay specifically or impinging also on either GATA or RUNX activity. Along this line, the results suggest that the three MED core modules but not the CDK8 regulatory module participate in Srp-induced transactivation. Importantly, too, 117 (90%) of the genes identified in the screen have well-conserved human homologs, suggesting they may regulate GATA/RUNX activity in humans. Actually, this sharp bias toward conserved genes underscores the fact that cell-based assays in Drosophila can serve as a powerful system to identify and characterize genes that may play similar roles in humans. Moreover, some homologs of Srp/Lz modifiers that were identified have been implicated in human diseases. These notably include MLF1, which is translocated in t(3;5)(q25.1;q34)-associated AML and whose Drosophila homolog is a target of Srp/Lz expressed in the crystal cells, as well as DDX10, which is translocated in inv(11)(p15q22)-associated AML. Whether these genes participate in GATA and/or RUNX function in normal or pathological situations in humans remains to be determined (Gobert, 2010).

The data show that the Mediator complex plays a central role in Srp/Lz-induced transactivation. Studies of yeast and metazoa highlighted the critical role of Mediator in both transcriptional activation and repression and showed that different Mediator subunits are required for the regulation of specific sets of genes or developmental processes. In addition, different transcription factors interact directly with specific Mediator subunits. Hence, the prevailing view is that different transcription factors depend on particular target proteins of the Mediator complex to regulate transcription. However, this study found that 20 of the 30 Mediator subunits were implicated as positive coregulators of Srp/Lz. Although some Mediator subunits, notably in the head module, play a global role in transcription, a general defect in transcription is unlikely to account for the observed decrease in Srp/Lz activity under the RNAi conditions, since no significant changes were observed in srp and Lz expression levels, except with Med19, a component of the head module, whose depletion decreased Lz levels. It is thus proposed that the integration of Srp/Lz transcriptional output requires the coordinated action of the different Mediator modules. However, it cannot be exclude that some of the Mediator subunits that were not identified in the screen may actually be dispensable for Srp/Lz activity (Gobert, 2010).

Remarkably, the CDK8 module, which is generally considered an accessory repressor module, was also required as a coactivator of Srp/Lz. Furthermore, in line with recent results revealing that all the functions of the CDK8 module do not rely on the CycC/Cdk8 pair, strong evidence is provided that only Med12/Med13 are required for the activation of Srp/Lz target genes in cell culture and in vivo. While different molecular mechanisms of repression by Cdk8/CycC and Med12/Med13 have been described, how Med12/Med13 may promote transcription remains elusive. These subunits may serve as an anchor to recruit the Mediator complex, as they have been shown to bind to Pygopus or ß-catenin to promote Wnt signaling and to Gli3 to inhibit Shh signaling. Accordingly, it was found that Srp and Lz interact with Med12 and Med13. However, this interaction could be due to another Mediator subunit required for Srp/Lz-induced transactivation. Alternatively, Med12/Med13 may be required for the proper folding of the Mediator complex to promote its interaction either with Srp/Lz or with downstream components of the transcriptional initiation machinery (Gobert, 2010).

In vivo, analysis of CDK8 module subunits shows that, reminiscent of what has been observed in larval imaginal discs, CycC/Cdk8 and Med12/Med13 have both common and specific functions during crystal cell development. Indeed, in the embryo, mutations in any of the four CDK8 module components resulted in a similar reduction in the absolute number of Lz+ blood cells and, concomitantly, of differentiated crystal cells, indicating that the whole CDK8 module controls the emergence of the crystal cell lineage. While the signaling that induces lz expression in the prohemocytes remains unknown, it was shown that the transcription factor Glial cell missing (Gcm) and the Friend of GATA corepressor U-shaped (Ush) oppose crystal cell fate choice. Both factors are expressed in the prohemocytes and interfere with lz expression to limit the number of crystal cells. Thus, loss of CDK8 module activity may impair crystal cell lineage emergence either by decreasing lz induction or by enhancing gcm or ush function (Gobert, 2010).

Similarly, it was found that targeted downregulation of Med12, CycC, or Cdk8 in the crystal cell lineage by RNAi after the onset of lz expression induced a cell-autonomous decrease in the absolute number of Lz+ larval blood cells. Hence, it is likely that the whole CDK8 module also controls the maintenance or the proliferation of the Lz+ cells during larval life. Recently, Wg signaling was shown to promote Lz+ larval blood cell proliferation. Interestingly, the CDK8 module participates in Wnt signaling. However, its coactivating function seemed to rely only on Med12/Med13 in Drosophila, whereas it depended on Cdk8/CycC in humans. Whether the CDK8 module regulates Lz+ cell number in response to Wg signaling or to another unknown pathway remains to be determined (Gobert, 2010).

In addition, the observation that only Med12 or Med13 downregulation caused a drop in the proportion of differentiated Lz+ cells in the larva strongly suggest that Med12/Med13 participates in crystal cell differentiation independently of the CycC/Cdk8 pair. In light of the results in cell culture, the in vivo data support an essential and direct function for Med12 and Med13 in the activation of the crystal cell differentiation program by Srp/Lz independently of CycC and Cdk8. All together, these data underline the functional flexibility of the CDK8 module, which appears to be reiteratively and specifically used at different stages of crystal cell development (Gobert, 2010).

In conclusion, it is anticipated that the results presented in this study lay the foundation for future investigations aiming at understanding the different levels of regulation of GATA and RUNX transcription factor activity not only in Drosophila but also in other species (Gobert, 2010).

Cyclin-dependent kinase 8 module expression profiling reveals requirement of mediator subunits 12 and 13 for transcription of Serpent-dependent innate immunity genes in Drosophila

The Cdk8 (cyclin-dependent kinase 8) module of Mediator integrates regulatory cues from transcription factors to RNA polymerase II. It consists of four subunits where Med12 and Med13 link Cdk8 and Cyclin C (CycC) to core Mediator. This study has investigated the contributions of the Cdk8 module subunits to transcriptional regulation using RNA interference in Drosophila cells. Genome-wide expression profiling demonstrated separation of Cdk8-CycC and Med12-Med13 profiles. However, transcriptional regulation by Cdk8-CycC was dependent on Med12-Med13. This observation also revealed that Cdk8-CycC and Med12-Med13 often have opposite transcriptional effects. Interestingly, Med12 and Med13 profiles overlapped significantly with that of the GATA factor Serpent. Accordingly, mutational analyses indicated that GATA sites are required for Med12-Med13 regulation of Serpent-dependent genes. Med12 and Med13 were also found to be required for Serpent-activated innate immunity genes in defense to bacterial infection. The results reveal a novel role for the Cdk8 module in Serpent-dependent transcription and innate immunity (Kuuluvainen, 2014).

In this genome-wide study on transcription regulation by the Cdk8 module, a striking pairwise similarity was noted between Cdk8 and CycC as well as Med12 and Med13. Co-regulation by all four subunits was surprisingly limited, clearly differing from yeast where depletion of any Cdk8 module subunit results in similar effects on transcription. Importantly, the lack of Med13-specific genes indicates that Med13 does not regulate transcription without Med12, although structural and biochemical analysis of the Cdk8 module suggests this might be possible. The results thus suggest that the previously identified Med13 regulatory mechanisms are likely to be directed toward functions of either Med12-Med13 or the entire Cdk8 module (Kuuluvainen, 2014).

The dependence of gene regulation by Cdk8-CycC on Med12-Med13 noted in this study and in a genetic study on leg bristles (Loncle, 2007) supports the suggested structural hierarchy where Cdk8-CycC is linked to the core Mediator through Med12 and Med13 (Adler, 2012; Chen, 2012). Identification of the dependence of Cdk8-CycC on Med12-Med13 also revealed that these pairs often have opposite transcriptional effects. This indicates that opposite regulation by Cdk8-CycC and Med12-Med13 should be considered as a possibility on all Cdk8-CycC-regulated genes and functions previously identified to be Med12-Med13-independent (Kuuluvainen, 2014).

Cdk8-CycC dependence on Med12-Med13 highlights the importance of investigating the possible involvement of Cdk8-CycC in Med12-Med13-dependent phenotypes. Interestingly, suppression of Shh signaling in cells with the FG and Lujan syndrome mutations in Med12 was recently shown to be a result of dissociation of Cdk8 but not Med12 on Gli3 target promoters (Zhou, 2012). Furthermore, the finding that Cdk8-CycC can act opposite to Med12-Med13 although being Med12-Med13-dependent indicates that, for example, loss of Med12 could lead to similar phenotypes as gain of Cdk8; both genetic alterations have been noted in human colorectal cancer. Taken together, these results are consistent with the notion that Cdk8-CycC mediates gene regulation primarily through interaction with Mediator through Med12-Med13, whereas Med12-Med13 can regulate transcription independently of Cdk8-CycC (Kuuluvainen, 2014).

Med12-Med13 was found in this study to be important for Srp-dependent transcription, and the previously identified physical interaction between Srp and Cdk8 module components provides a plausible mechanism for this. In addition to Mtk and DptB, the IMD target CecA1 is also a target of Srp and dependent on the Cdk8 module components. Consistent with this, induction of the A. gambiae homolog of CecA1, Cec1, was recently shown to require Med12 and Med13. Multiple known (e.g., Eater, Sr-CI, Pxn) and novel (e.g., CG14629, CG10962) Srp-dependent genes found in this study to be Med12-Med13-dependent implicate Med12-Med13 in various Srp-regulated functions. Besides its role in AMP gene induction, Srp is required in hematopoietic differentiation. In some instances, this may be modulated by the Cdk8 module, suggested by the requirement of Drosophila Med12-Med13 and zebrafish Med12 in differentiation of specific blood cell lineages. Based on this, it will be interesting to study the possible involvement of Med12-Med13 in mammalian GATA-dependent hematopoiesis (Kuuluvainen, 2014).

It appears that transcription regulation by the Cdk8 module is largely context-dependent. In this regard, it was intriguing to identify several genes involved in neuronal functions (Epac, Fie, ogre, and PQBP-1) as strongly regulated by Med12-Med13 in S2 cells. It will be interesting to analyze whether these genes are also regulated by Med12-Med13 in neural tissues, where Med12 and one of it targets identified here, PQBP-1, present related phenotypes (Kuuluvainen, 2014).

cis-regulatory requirements for tissue-specific programs of the circadian clock

Broadly expressed transcriptions factors (TFs) control tissue-specific programs of gene expression through interactions with local TF networks. A prime example is the circadian clock: although the conserved TFs Clock (Clk) and Cycle (Cyc) control a transcriptional circuit throughout animal bodies, rhythms in behavior and physiology are generated tissue specifically. Yet, how Clk and Cyc determine tissue-specific clock programs has remained unclear. This study used a functional genomics approach to determine the cis-regulatory requirements for clock specificity. First Clk and Cyc genome-wide binding targets in heads and bodies were determined by ChIP-seq, and they were shown to have distinct DNA targets in the two tissue contexts. Computational dissection of Clk/Cyc context-specific binding sites reveals sequence motifs for putative partner factors, which are predictive for individual binding sites. Among them, it was shown that the opa and GATA motifs, differentially enriched in head and body binding sites respectively, can be bound by Opa and Serpent (Srp). They act synergistically with Clk/Cyc in the Drosophila feedback loop, suggesting that they help to determine their direct targets and therefore orchestrate tissue-specific clock outputs. In addition, using in vivo transgenic assays, it was validated that GATA motifs are required for proper tissue-specific gene expression in the adult fat body, midgut, and Malpighian tubules, revealing a cis-regulatory signature for enhancers of the peripheral circadian clock. These results reveal how universal clock circuits can regulate tissue-specific rhythms and, more generally, provide insights into the mechanism by which universal TFs can be modulated to drive tissue-specific programs of gene expression (Meireles-Filho, 2013).

Although frequently not restricted to single cell types, individual TFs can control tissue-specific programs of gene expression through interactions with local TF networks. But despite substantial progress in identifying differential cell-specific circadian expression programs, how Clk and Cyc interact with local TF networks to generate output rhythms tissue specifically is still elusive (Meireles-Filho, 2013).

This study used an integrative genomics approach to shed light on how the circadian clock drives tissue-specific gene expression. While shared Clk/Cyc binding sites could not be explained by combinations of head- and body-specific motifs, yet were slightly more enriched in E box motifs and -- similar to highly occupied target [HOT] regions -- in Trithorax-like motifs [Trl/GAGA; 2-fold]), a substantial number of Clk and Cyc binding sites were specific to either heads or bodies and next to genes with different functional GO categories. These binding sites differed substantially in their motif content, and this motif signature was predictive of context-specific Clk/Cyc binding, suggesting that tissue-specific clock targets are determined by the binding site sequences (Meireles-Filho, 2013).

GATA motifs were enriched in Clk/Cyc binding sites in bodies and required for enhancer activity in the fat body, midgut, and Malpighian tubules. This suggests that GATA factors might play a key role for Clk/Cyc-bound enhancers in bodies, potentially by helping to establish the chromatin landscape in tissues where they are specifically expressed (e.g., srp in the fat body and GATAe in the gut). Interestingly, GATA motifs are also overrepresented in promoter regions of circadian genes in rodents, suggesting a conserved role for GATA factors in the circadian clock (Meireles-Filho, 2013).

This study found that the GATA factor Srp could act synergistically with Clk, suggesting that it is an important determinant of clock function in peripheral tissues. Srp has multiple functions in Drosophila, including the control of endodermal development and hematopoiesis in the embryo and the induction of immune response in the larval fat body. Interestingly, srp is coexpressed with Clk and Cyc in the fat body, a tissue with roles in metabolic activity, innate immunity response, and detoxification - all known to be controlled in a circadian manner. Clk body-specific peaks were 4.17-fold enriched close to cycling fat body genes, suggesting that srp might help determine the physiological outputs controlled by the fat body pacemaker. Interestingly, srp is also required for hormone-induced expression of the Fbp1 TF during fat body development, supporting the idea that it might be important for temporal or inducible regulation more generally (Meireles-Filho, 2013).

Similarly, Opa, which belongs to the Zic family of mammalian TFs with conserved roles in head formation in flies and mammals, is coexpressed with Clk and cyc in the adult brain. In addition, an enhancer of Slob, an output gene of the clock pacemaker involved in the generation of locomotor activity rhythms, responded to Clk and Cyc in an Opa-dependent manner, suggesting that Opa might be involved in the recruitment of Clk/Cyc to regulate genes controlling fly behavior. Further studies on Opa and additional predicted partner TFs might provide new insights into the Drosophila clock in the head (Meireles-Filho, 2013).

It is likely that different cofactors with functions equivalent to srp or opa exist in different cell types, which redirect Clk/ Cyc to tissue-specific binding sites and allow tissue-specific gene regulation. Indeed, this study has identified several other motifs that are tissue-specifically enriched. This is reminiscent of studies showing that TFs downstream of signaling pathways are redirected in a tissue-specific manner by cell-specific master regulators. The results might thus constitute an important example of how partner TFs adapt broadly active transcriptional regulators to achieve tissue-specific gene expression and function, contributing to a better understanding of gene regulatory networks more generally (Meireles-Filho, 2013).

These data on Clk/Cyc binding in different contexts not only provide novel insights into clock regulatory networks and enhancer structure but also exemplify a new strategy to uncover cofactors of the circadian clock via their cis-regulatory motifs. This approach is complementary to forward and reverse genetics or biochemistry, which have traditionally been used to reveal clock factors. It can also be applied more generally to identify factors that recruit broadly expressed TFs in different cell types or tissues. In addition, the tagging of endogenous loci allows the study of TFs under physiological conditions in their endogenous expression domains, which is crucial especially for TFs that have large and complex regulatory regions and/or for which physiological expression levels are of fundamental importance. In summary, the results in the Drosophila circadian clock reveal how universal TF circuits can be modulated to generate transcriptional tissue-specific outputs and demonstrate a novel approach to determine regulatory partners more generally (Meireles-Filho, 2013).

Protein Interactions

Friend of GATA (FOG) proteins regulate GATA factor-activated gene transcription. During vertebrate hematopoiesis, FOG and GATA proteins cooperate to promote erythrocyte and megakaryocyte differentiation. The Drosophila FOG homolog U-shaped (Ush) is expressed similarly in the blood cell anlage during embryogenesis. During hematopoiesis, the acute myeloid leukemia 1 homolog Lozenge and Glial cells missing are required for the production of crystal cells and plasmatocytes, respectively. However, additional factors have been predicted to control crystal cell proliferation. Ush is expressed in hemocyte precursors and plasmatocytes throughout embryogenesis and larval development, and the GATA factor Serpent is essential for Ush embryonic expression. Furthermore, loss of ush function results in an overproduction of crystal cells, whereas forced expression of Ush reduces this cell population. Murine FOG-1 and FOG-2 also can repress crystal cell production, but a mutant version of FOG-2 lacking a conserved motif that binds the corepressor C-terminal binding protein fails to affect the cell lineage. The GATA factor Pannier (Pnr) is required for eye and heart development in Drosophila. When Ush, FOG-1, FOG-2, or mutant FOG-2 is coexpressed with Pnr during these developmental processes, severe eye and heart phenotypes result, consistent with a conserved negative regulation of Pnr function. These results indicate that the fly and mouse FOG proteins function similarly in three distinct cellular contexts in Drosophila, but may use different mechanisms to regulate genetic events in blood vs. cardial or eye cell lineages (Fossett, 2001).

Srp function is required for hemocyte development and for differentiation of plasmatocytes and crystal cells. Furthermore, studies using amorphic alleles of srp indicate that it is required for hemocyte precursor specification. Srp is expressed first in the hemocyte precursors during embryonic stage 5, and, similar to Ush, its expression is maintained in plasmatocytes throughout embryogenesis. To determine whether an epistatic relationship exists between srp and ush, Ush expression was assayed in srp mutant embryos and Srp expression in ush mutant embryos. The hypomorphic allele srp3, which results in the production of hemocyte precursors, even with the reduction of Srp function, was used. In srp embryos, Ush is not detected in hemocyte precursors, plasmatocytes, or midgut, unlike the wild-type expression pattern. In contrast, Srp is observed in hemocyte precursors and plasmatocytes in both wild-type and ush mutant embryos. This result suggests ush resides downstream of srp in the hematopoiesis hierarchy and ush expression requires Srp function. Furthermore, ush is not required for the specification of hemocyte precursors or plasmatocytes, because these Srp-positive cells are detected in ush mutant embryos. Finally, wild-type levels of ush are present in the dorsal ectoderm of srp mutant embryos, indicating that dynamic ush expression is under the control of multiple regulators during embryogenesis (Fossett, 2001).

FOG function involves binding to its GATA partner's N-terminal zinc finger. Srp is the only known hematopoietic GATA factor in Drosophila and reportedly contains a single C-terminal zinc finger. However, a survey of the srp genomic sequence shows an ORF within the third intron of the gene that putatively encodes an N-terminal zinc finger with 96% homology to that of Pnr. This raises the possibility that Ush interacts with an alternatively spliced isoform of Srp during hematopoiesis (Fossett, 2001).

serpent encodes a GATA transcription factor essential for hematopoiesis in Drosophila. Previously, Srp was shown to contain a single GATA zinc finger of C-terminal type. srp encodes different isoforms, generated by alternative splicing, that contain either only a C-finger (SrpC) or both a C- and an N-finger (SrpNC). The presence of the N-finger stabilizes the interaction of Srp with palindromic GATA sites and allows interaction with the Friend of GATA factor U-shaped (Ush). The respective functions of SrpC and SrpNC during embryonic hematopoiesis were examined. Both isoforms individually rescue blood cell formation, which is lacking in a srp null mutation. Interestingly, while SrpC and SrpNC activate some genes in a similar manner, they regulate others differently. Interaction between SrpNC and Ush is responsible for some but not all aspects of the distinct activities of SrpC and SrpNC. These results suggest that the inclusion or exclusion of the N-finger in the naturally occurring isoforms of Srp can provide an effective means of extending the versatility of srp function during development (Waltzer, 2002).

In a systematic search for GATA zinc finger-coding sequences in the Drosophila genome, five genes were found: dGATA-E (CG10278), dGATA-D (CG5034), pnr, grain and srp. dGATA-E and dGATA-D appear to include only a C-finger, while Pnr and Grain contain both an N- and a C-finger. Interestingly, while Srp has been reported to contain a single C-finger, the presence of a putative exon (E4A) coding for an N-finger motif in srp is also evident. Using RT–PCR assays with various combinations of oligonucleotides, it has been shown that E4A is expressed and that E4A and E4B are alternatively spliced to exon 5. In the course of these experiments, an additional splice acceptor site was also identified within E7. This downstream acceptor site in E7 is out-of-frame and leads to the deletion of the Srp glutamine-rich C-terminal region. The data indicate that four alternatively spliced mRNAs are transcribed from srp, two encoding products with a single C-finger (SrpC and SrpCd) and two encoding products with both N- and C-fingers (SrpNC and SrpNCd). Interestingly, in SrpNC and SrpNCd, the two fingers present the same conserved organization as in other GATA factors. Notably, they are separated by 29 amino acids, as in all vertebrate GATA. The two isoforms that contain the full-length exon 7, i.e. srpC and srpNC, have been used to address the functional consequences of the alternative splicing of E4A and E4B (Waltzer, 2002).

Whether SrpC and SrpNC display different properties in vitro was investigated. While the C-finger is necessary and sufficient for specific DNA binding, it has also been shown in vertebrates that the N-finger can stabilize the binding to particular double GATA sites. By electophoretic mobility shift assays (EMSAs), it was determined if SrpNC and SrpC have similar DNA-binding properties. Both in vitro translated SrpC and SrpNC proteins bind to an oligonucleotide containing a consensus GATA site. The binding is specific, since it can be competed out efficiently by an excess of cold GATA oligonucleotide, but not by an excess of the GATC oligonucleotide. The stability of the SrpN and SrpNC complex on a single or on a palindromic GATA site was assessed by dissociation experiments. While the rate of dissociation is similar for SrpC and SrpNC on a single GATA probe, SrpNC bound more stably than SrpC to the palindromic GATA sites (Waltzer, 2002).

The GATA N-finger allows interaction with cofactors of the FOG family. Key residues that are required for the interaction between GATA and FOG are conserved in the Srp N-finger. In order to test the binding between Ush and srp products, pull-down assays were performed in vitro. in vitro translated [35S]methionine-labelled Ush binds to GST–SrpNC, but not GST–SrpC. Thus, Ush specifically interacts with Srp isoforms that contain the N-finger. In addition, like its vertebrate homologs, Ush interacted with the transcriptional corepressor dCtBP in this assay (Waltzer, 2002).

Taken together, the results indicate that SrpNC displays features characteristic of two-fingered GATA factors. The two types of naturally occurring isoforms encoded by srp (with or without the N-finger) have different DNA-binding properties, and only the isoforms including an N-finger can interact with Ush (Waltzer, 2002).

In order to determine whether a spatial regulation of the alternative splicing leading to SrpC and SrpNC occurs during embryonic development, the distribution of the corresponding srp transcripts was assessed by in situ hybridization using specific probes for exon 4A or 4B. At the blastoderm stage and during gastrulation, srpC and srpNC show the same expression pattern. They are expressed in the procephalic mesoderm, the hemocyte primordium, at the anterior and posterior pole, in the primordium of the anterior and posterior midgut as well as in the amnioserosa and in the yolk cells. Later, during germ band extension, and after germ band retraction, srpC and srpNC are expressed identically in the developing fat body. Thus, srpC and srpNC transcripts are not differentially regulated spatially during embryonic development. However, the level of the transcripts is not identical. Indeed, by means of semi-quantitative RT–PCR, it was determined that exon 4B-containing mRNA is five times more abundant than exon 4A-containing mRNA, suggesting that two-fingered isoforms of Srp are less abundant than single-fingered isoforms (Waltzer, 2002).

In order to analyse SrpC and SrpNC activities, their capacities to activate gene expression in vivo were tested during Drosophila embryonic hematopoiesis. Using the UAS-GAL4 system, they were ectopically expressed in the mesoderm and the expression pattern of various hematopoietic markers was assessed. The two genes ush and gcm play critical roles in embryonic hematopoiesis. Their expression in the hematopoietic primordium occurs early and appears to depend on srp activity. Therefore, it was determined whether they are transcriptional targets of SrpC and/or SrpNC. Whereas in a wild-type early embryo, ush expression is restricted to the anterior mesoderm, twist-driven expression of SrpC (twist-SrpC) or SrpNC (twist-SrpNC) induces strong expression of ush throughout the mesoderm. In contrast, twist-SrpC induces gcm expression poorly and in a limited number of mesodermal cells of stage 5 embryos, whereas twist-SrpNC strongly activates gcm expression segmentally from stage 5 to 9 (Waltzer, 2002).

The expression of hematopoietic lineage-specific markers was examined. As plasmatocyte markers, peroxidasin (pxn) and croquemort (crq) were used. Since, crystal cells are the only source of prophenoloxidase (pro-PO) in Drosophila, expression of this gene was used to monitor crystal cell formation. pro-PO transcripts were indeed detected in these cells from early stage 11 to the end of embryogenesis. Analysing these markers, two situations were observed. twist-SrpC and twist- SrpNC have similar abilities to induce expression of the plasmatocyte marker pxn and of the crystal cell marker pro-PO, however expression of crq was induced by twist-SrpC and not by twist-SrpNC. Note that pxn and crq were induced through most of the mesoderm, while pro-PO activation was restricted to the head region (Waltzer, 2002). Taken together, these data show that SrpC and SrpNC have both common and different activities during hematopoiesis. Indeed, both isoforms activate the expression of ush, pxn and pro-PO in a similar manner. However, SrpC and SrpNC differentially stimulate the expression of crq and gcm, respectively, in the mesoderm (Waltzer, 2002).

It is remarkable that srp encodes both single and dual zinc finger-containing products. The results provide strong evidence that this alternative splicing allows production of transcription factors with specific activities. The two isoforms activate the expression of ush and pxn with similar efficiency, suggesting that SrpC and SrpNC have similar transactivating properties in vivo, yet, SrpC (but not SrpNC) activates crq expression, while SrpNC is a much stronger activator of gcm expression than SrpC. The domain coded by exon 4B that is present only in SrpC has no known motif and it is not known if and how it participates in SrpC-specific function. However, the presence of the N-terminal zinc finger encoded by exon 4A may explain some of the distinct features of SrpNC as discussed below (Waltzer, 2002).

As in the case of vertebrate GATA-1, the presence of the N-finger in Srp stabilizes binding to double palindromic GATA sites. Although the N-finger of GATA-1 modulates the binding and the transactivating properties of GATA-1 on synthetic promoters, the functional importance of these effects has remained elusive, particularly since no GATA-1 isoform contains only the C-finger. In the case of srp, these distinct binding properties may have direct functional consequences. For instance, the fact that SrpC and SrpNC activate a common target, ush, whereas only SrpNC strongly activates a specific target, gcm, could be related to the DNA-binding specificity of the two isoforms. A scan of the ush upstream regulatory region shows that it contains several GATA consensus sequences, nine of which are clustered in <1 kb and are organized as three repetitions of three sites. In contrast, GATA sites are far less frequent in gcm regulatory regions and are often organized in palindromes. Considering that ush and gcm are likely to be direct target genes for srp, the different organization of their regulatory regions may explain the differential effect observed (Waltzer, 2002).

The lack of plasmatocyte and crystal cell formation due to an srp null mutation can be rescued by expressing SrpC or SrpNC in the mesoderm. No difference between the two isoforms was seen in this assay, suggesting that the N-finger is not absolutely required for srp function in embryonic blood cell formation. However, in the absence of a functional test, to what extent the formation of embryonic blood cells is fully rescued cannot be determined. Interestingly, rescue experiments with the mouse GATA-1 mutant indicate that the GATA-1 N-finger is dispensable for primitive erythropoiesis but is required for definitive erythopoiesis. In Drosophila, a second wave of hematopoiesis, occurring at the larval stage, gives rise to four different lineages: plasmatocytes, crystal cells, secretory cells and lamellocytes. srp is expressed in the dorsal lymph gland (i.e. the main larval hematopoietic organ) and it probably controls larval hematopoiesis. By analogy to vertebrate GATA-1, the Srp N-finger may provide an additional function for larval hematopoiesis, perhaps during formation of the new cell types (Waltzer, 2002).

In the assay used, the expression of the transgene was limited to the mesoderm but it still rescued blood cell formation. This finding suggests that the early expression of srp in the hematopoietic primordium is sufficient to initiate the genetic program that controls hemocyte formation and differentiation. Interestingly, in the wild-type embryo, srp transcripts are not expressed detectably in hemocytes after stage 11, but Srp protein is detected in plasmatocytes and crystal cells throughout most of embryogenesis. Persistence of srp products in hemocytes might be critical for srp function, and control of srp products at the post-translational level may play a crucial role in the correct regulation of blood cell differentiation. Rescue of crystal cell formation by mesodermal expression of SrpC and SrpNC contrasts with the observation that later expression driven by lz-Gal4 in crystal cells represses their development. Srp levels are reduced in crystal cells compared with surrounding plasmatocytes. Therefore, the results are consistent with a two-step model in which Srp expression is first necessary to induce lz expression and subsequently is downregulated to allow crystal cell differentiation (Waltzer, 2002).

One of the best characterized features of GATA N-fingers is their dimerization with cofactors of the FOG family. Consistent with this feature, it was found that SrpNC interacts with the Drosophila FOG Ush, but SrpC does not. Previous analysis showed that ush regulates the number of crystal cells. It was proposed that this function of ush could be mediated by a putative isoform of Srp containing an N-finger. The current findings strongly support this hypothesis. However, it was not possible to address this issue directly, since both SrpC and SrpNC display a strong Ush-independent repressive effect on crystal cell formation and differentiation (Waltzer, 2002).

A new function of ush revealed here is the regulation of the level of expression of the macrophage receptor crq, suggesting that ush displays a broader function in hematopoiesis than previously assumed. Notably, evidence is provided that Ush modulates SrpNC transactivation of crq. Since Ush interacts with the corepressor dCtBP in vitro, the Ush–SrpNC complex could repress crq expression. However, it is not known whether crq is a direct target of srp, so the possibility that the Ush–SrpNC complex activates a transcriptional repressor that regulates crq cannot be ruled out. Vertebrate FOGs can act as either a coactivator or a corepressor of GATA factors. In Drosophila, Ush is a repressor of Pannier-induced activation in cell culture, and it probably also represses the expression of achaete in the dorso-central proneural cluster in vivo. Furthermore, in a heterologous assay in Drosophila, the CtBP-binding region of mFOG2 is required for repressing the formation of crystal cells but not cardiac cells. Thus several mechanisms seem to regulate the function of the GATA–FOG complex (Waltzer, 2002).

Remarkably, some functions of SrpNC appear to be independent of Ush. Thus, gcm-specific activation by SrpNC is not affected in an ush mutant embryo. Moreover, SrpNC still represses crystal cell formation in the absence of ush. This is reminiscent of mouse erythropoiesis, where both FOG-dependent and FOG-independent regulation of gene expression by GATA-1 have been observed. The molecular mechanisms underlying the regulation by Ush/FOG-1 of SrpNC/GATA-1 activity on some specific targets remain to be elucidated. It is tempting to speculate that the N-finger of SrpNC is involved in the recognition of promoter sequences, on gcm for example, and thus is not available to recruit Ush. Alternatively, other cofactors already localized to the promoter or bound to SrpNC might prevent Ush binding to the N-finger (Waltzer, 2002).

This study has focussed on hematopoiesis, but srp also participates in other developmental processes, such as germ band retraction, midgut differentiation, fat body formation, induction of the immune response and the ecdysone response. It will be interesting to determine the respective roles of SrpC and SrpNC in these different phenomena. Phylogenetic analysis shows that SrpNC is closely related to vertebrate GATA factors. It has been suggested that srp is a functional homolog of the entire vertebrate GATA family, since srp is required in Drosophila for hematopoiesis, like GATA-1/2/3 in mice, and for endodermal development, like GATA-4/5/6. Nevertheless, this hypothesis was at odds with the fact that Srp seemingly had a single zinc finger while all the vertebrate GATAs have two. The present identification of Srp isoforms with two fingers gives new force to this hypothesis. Further, the expression of isoforms of Srp with distinct activities helps to account for the broad range of functions ensured by this gene (Waltzer, 2002).

It is worth noting that alternative splicing eliminating the N-finger has also been described in Bombyx mori GATAß and in chicken GATA-5 genes. Moreover, a BLAST search analysis revealed alternatively spliced human expressed sequence tags coding for two isoforms of a potential GATA factor with either one or two zinc fingers. This suggests that alternative splicing of GATA genes could be more general than previously thought, and as yet unnoticed splice variants of GATA vertebrate genes may generate proteins with only a C-finger (Waltzer, 2002).

In conclusion, these results shed further light on the molecular control of hematopoiesis by the GATA factor Srp. The alternative splicing of srp gives rise to different Ush-interacting and non-interacting Srp proteins with different target gene specificities, thereby contributing to the exquisite control of Drosophila blood cell formation. It is speculated that alternative splicing of the GATA N-finger might be an important mechanism regulating the activity of other GATA genes from insects to man (Waltzer, 2002).

The Friend of GATA protein U-shaped functions as a hematopoietic tumor suppressor in Drosophila

Drosophila has emerged as an important model system to discover and analyze genes controlling hematopoiesis. One regulatory network known to control hemocyte differentiation is the Janus kinase (JAK)/Signal Transducer and Activator of Transcription (STAT) signal-transduction pathway. A constitutive activation mutation of the Janus kinase Hopscotch (hopscotchTumorous-lethal; hopTum-l) results in a leukemia-like over-proliferation of hemocytes and copious differentiation of lamellocytes during larval stages. Friend of GATA (FOG) protein U-shaped (Ush) is expressed in circulating and lymph gland hemocytes, where it plays a critical role in controlling blood cell proliferation and differentiation. These findings demonstrate that a reduction in ush function results in hematopoietic phenotypes strikingly similar to those observed in hopTum-l animals. These include lymph gland hypertrophy, increased circulating hemocyte concentration, and abundant production of lamellocytes. Forced expression of N-terminal truncated versions of Ush likewise leads to larvae with severe hematopoietic anomalies. In contrast, expression of wild-type Ush results in a strong suppression of hopTum-l phenotypes. Taken together, these findings demonstrate that U-shaped acts to control larval hemocyte proliferation and suppress lamellocyte differentiation, likely regulating hematopoietic events downstream of Hop kinase activity. Such functions appear to be facilitated through Ush interaction with the hematopoietic GATA factor Serpent (Srp) (Sorrentino, 2007).

In wild-type lymph glands, Ush is not detectable in the second instar larva (L2) but is expressed in L3, beginning in the cortical zone and eventually spreading to the entire lymph gland. It stands to reason that during the normal dispersal of the hematopoietic organs in late L3, those lymph gland hemocytes in the cortical zone will be the first to enter circulation. In such a model, Ush-expressing lymph gland hemocytes enter the circulating hemocyte population, which (with the exception of crystal cells) already express Ush. Thus Ush can be viewed as a hemocyte maturation marker. This begs the question of why Ush is expressed in what are apparently the most mature hemocytes. The current observations strongly implicate Ush as being present in order to suppress proliferation and lamellocyte differentiation among mature plasmatocytes (Sorrentino, 2007).

The strongest evidence for a mechanism in which Ush suppresses hemocyte proliferation and lamellocyte differentiation comes from analyses of ush mutants. Reducing Ush function causes lymph gland hypertrophy, which is a direct result of an increase in the number of lymph gland hemocytes. Furthermore, in a manner similar in quality (but somewhat less in intensity) to those of hopTum-l larvae, ush mutant lymph glands disperse precociously, and cortical zone hemocytes appear to be morphologically consistent with lamellocytes. Additionally, total circulating hemocyte concentration (CHC) of ush mutants is over four-fold greater than that of wild-type larvae, and two different alleles of ush when heterozygous, induce a less severe but nonetheless significant hematopoietic phenotype. High CHC is consistent with the mechanism of a large number of hemocytes, including lamellocytes, leaving the lymph gland and entering circulation. The possibility cannot be ruled out that circulating hemocytes, which are of a different embryonic origin than lymph gland hemocytes, can also over-proliferate and/or differentiate into lamellocytes in ush mutants. Since cortical zone hemocytes (predominantly plasmatocytes) express Ush and can differentiate into lamellocytes, the fact that circulating plasmatocytes also express Ush would support the notion of circulating plasmatocytes also being able to differentiate into lamellocytes (Sorrentino, 2007).

Importantly, it was observed that wild-type L2 lymph glands do not express Ush, while L3 organs do (in the cortical zone first, then throughout the lymph gland). Clearly, a mechanism that represses a developmental decision is not necessary unless a cell has the potential to actually make the choice. Thus the existence of a Ush-regulated mechanism for suppression of hemocyte proliferation and lamellocyte differentiation in L3 cortical zone hemocytes is interpreted as supportive of the hypothesis that Ush+ cells are in a different genetic state in which they can, given the proper cues, hyperproliferate and differentiate into lamellocytes. It follows that wild-type L2 lymph gland hemocytes cannot hyperproliferate and become lamellocytes. Such a putative mechanism is consistent with previous findings; Jung (2005) observed that L3 lymph gland hemocyte proliferation takes place primarily within the cortical zone, while Sorrentino (2002) observed that, in larvae parasitized by the wasp Leptopilina boulardi, L2 lymph glands are immune-unresponsive (as indicated by mitotic index, crystal cell population size, and a lamellocyte marker) whereas L3 lymph glands do respond (Sorrentino, 2007).

Additional strong evidence for the role of Ush is provided by transgene expression data. Expression of wild-type Ush in hopTum-l/Y larvae produced an effect opposite in quality to that of ushVX22/ushr24, that being a significant 90% reduction in the hopTum-l-induced circulating lamellocyte population. The CgGAL4 driver is active in hopTum-l/Y L2 hemocytes, thus transgenic Ush has an opportunity to act on hemocytes prior to lymph gland dispersal. The significant reduction could be explained by the suppression of lamellocyte differentiation and/or the suppression of the proliferation of pro-lamellocytes. An apoptotic mechanism may also be partially involved. The reason for the observation that the hopTum-l non-lamellocyte population was not significantly affected by transgenic Ush is unknown, but one explanation would be a dosage-dependent mechanism in which experimental expression of just one copy of a UASush transgene is insufficient to suppress hopTum-l over-proliferation. It is also possible that hemocyte over-proliferation is a secondary effect of lamellocyte differentiation, and thus not under the direct control of Ush (Sorrentino, 2007).

If Ush normally suppresses crystal cell differentiation why do ush mutants not exhibit a severe overabundance of crystal cells? Using the strong amorphic ush1 background, an approximately 30% increase in mean crystal cell counts has been observed in stage-16 embryos. However, since the wild-type mean number of crystal cells in stage-16 embryos is about 24-25 per embryo, a 30% increase amounts to about 8 additional crystal cells. Since there are hundreds of plasmatocytes in an embryo, the overwhelming majority of plasmatocytes do not become crystal cells in the absence of Ush (Sorrentino, 2007).

Such findings can be explained by a model in which Ush suppresses crystal cell differentiation in a small subset of hemocytes, with the primary role of Ush in hematopoiesis being to control lamellocyte differentiation and hemocyte proliferation. In this model, the down-regulation of ush expression in embryonic crystal cells occurs because hemocytes committed to the crystal cell lineage cannot become lamellocytes, and thus require no mechanism to suppress lamellocyte differentiation (Sorrentino, 2007).

Srp is expressed in all hopTum-l/Y hemocytes, both lamellocytes and non-lamellocytes, in circulation and in the lymph gland. Thus, all Drosophila hemocyte classes studied thus far express this hematopoietic GATA factor, and the role of Srp in the differentiation of all hemocyte types is worthy of investigation. The fact that the lamellocyte population observed in ushVX22/+ larvae is strongly reduced by srpneo45/+ and completely reduced to wild-type levels by srp3/+ suggests Srp plays an active role in lamellocyte differentiation. In humans, GATA-3 determines the differentiation of Th2 cells, which like lamellocytes are the primary effectors in a cellular immune response against metazoan endoparasites. FOG-1 inhibits Th2 differentiation by inhibiting GATA-3 activity via physical interaction. However, if Srp is indeed necessary for lamellocyte differentiation, the finding that Srp is expressed in all hemocytes likely means it is not the sole determinant of lamellocyte differentiation. Thus it is considered likely that an additional transcriptional regulator, either another GATA factor (e.g., Grain, dGATAd, dGATAe) or a non-GATA factor, works in conjunction with Srp to specify the lamellocyte differentiation program (Sorrentino, 2007).

Ush232-1191, Ush302-1191, and Ush365-1191 driven by CgGAL4 are dominant inducers of hematopoietic tumor phenotypes, measurably stronger than that of the ushVX22/ushr24 loss-of-function condition. This finding is not interpreted as coincidental. In an important parallel all three of these constructs, when activated by the mesodermal twiGAL4 driver, exhibit a failure to suppress the expression of a cardiac-active Dmef2-lacZ reporter gene. Additionally, it was observed that Ush232-1191, though missing zinc finger 1, is still able to bind to Srp in vitro just as it is able to bind to Pnr. Such observations are consistent with the possibility that endogenous and transgenic Ush may compete in their binding to Srp. In such a situation, transgenic Ush232-1191, even if bound to Srp, might fail to suppress Srp-induced hemocyte proliferation and lamellocyte differentiation. Alternative explanations include: (1) Ush232-1191 bound to Srp may actually enhance normal Srp activity; (2) the Srp:Ush232-1191 complex may behave neomorphically; (3) Ush may normally dimerize while not bound to Srp, if so a Ush:Ush232-1191 complex may not be able to separate into active Ush monomers. The transgenic Ush proteins are assumed to be sufficiently stable and functional as to validate these observations, since the UASush constructs used have been shown to generate stable proteins in other cell types and also to induce measurable phenotypes (Sorrentino, 2007).

There is significantly more Ush present in the nuclei of hopTum-l/Y hemocytes than in nuclei of wild-type blood cells. Interestingly, Ush appears to be exclusively nuclear in hopTum-l/Y hemocytes, whereas there appears to be some cytoplasmic anti-Ush staining in wild-type hemocytes. It is possible that Ush function is in part determined by its cytoplasmic/nuclear ratio. Perhaps the qualitatively higher Ush concentration in hopTum-l hemocytes is the result of nuclear translocation of all cellular Ush. Based on work with human 293T and mouse erythroleukemia cell cultures, Garriga-Canut (2004) proposed a model in which TACC-3 and GATA-1 compete in binding to FOG-1, with FOG-1 bound to TACC-3 retained in the cytoplasm. Such a mechanism may also be at work in Drosophila hemocytes and the possibility of a Ush cytoplasmic sequestration phenomenon remains to be investigated (Sorrentino, 2007).

This study also found that there exist high concentrations of exclusively nuclear Ush in other tumorous backgrounds, those being in Tl10b/+ and CgGAL4>UAScol animals. Larvae carrying the dominant Tl10b allele exhibit a hematopoietic tumor phenotype similar to that of hopTum-l/Y larvae. In addition to srpDGAL4>UAScol, CgGAL4>UAScol is sufficient to induce lamellocyte differentiation (although it also induces L2 developmental arrest). All hemocytes, including lamellocytes, in both of these backgrounds also exhibit high concentrations of nuclear Ush. These observations are consistent with a model in which three different signaling pathways (Hop-Stat, Toll-Dorsal, and the early B-cell related factor Collier) all make use of a single common downstream lamellocyte induction program that involves Ush. Examination of hemocytes from additional tumorous backgrounds will reveal whether such a model is truly universal. An important question remains as to how Ush suppresses hemocyte proliferation and lamellocyte differentiation, yet is expressed so strongly in tumorous hemocytes. Taken together, the findings are supportive of Ush having an early function in repressing lamellocyte differentiation. Up-regulation of the protein in lamellocytes would be suggestive of a second, separate function for Ush within this differentiated hemocyte. Comparable multi-functional properties have been reported for FOG-1 in vertebrate hematopoiesis (Sorrentino, 2007).

Therefore, reduction of Ush function results in a classic hematopoietic tumor phenotype: lymph gland hypertrophy and early dispersal, a significant increase in total circulating hemocyte concentration, large-scale lamellocyte differentiation, and melanotic tumors. These anomalies can be partially induced by the loss-of-function of a single copy of ush. The identification of this FOG class protein as a tumor suppressor raises questions about the roles of other FOG proteins in mammalian leukemias. While mutations in murine fog1 have been associated with hematopoietic dysfunction such as the failure of megakaryopoiesis and the arrest of erythropoiesis, FOG proteins have not been implicated as a causal factor in any human leukemia. While there is no guarantee that the observations in Drosophila will directly translate to specific human hematopoietic pathologies, it may now be worthwhile to examine the state of fog gene expression and function in human leukemias (Sorrentino, 2007).

serpent: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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